Proteins, Small Peptides and Other Signaling Molecules Identified as Inconspicuous but Possibly Important Players in Microspores Reprogramming Toward Embryogenesis

In this review, we describe and integrate the latest knowledge on the signaling role of proteins and peptides in the stress-induced microspore embryogenesis (ME) in some crop plants with agricultural importance (i.e., oilseed rape, tobacco, barley, wheat, rice, triticale, rye). Based on the results received from the most advanced omix analyses, we have selected some inconspicuous but possibly important players in microspores reprogramming toward embryogenic development. We provide an overview of the roles and downstream effect of stress-related proteins (e.g., β-1,3-glucanases, chitinases) and small signaling peptides, especially cysteine—(e.g., glutathione, γ-thionins, rapid alkalinization factor, lipid transfer, phytosulfokine) and glycine-rich peptides and other proteins (e.g., fasciclin-like arabinogalactan protein) on acclimation ability of microspores and the cell wall reconstruction in a context of ME induction and haploids/doubled haploids (DHs) production. Application of these molecules, stimulating the induction and proper development of embryo-like structures and green plant regeneration, brings significant improvement of the effectiveness of DHs procedures and could result in its wider incorporation on a commercial scale. Recent advances in the design and construction of synthetic peptides–mainly cysteine-rich peptides and their derivatives–have accelerated the development of new DNA-free genome-editing techniques. These new systems are evolving incredibly fast and soon will find application in many areas of plant science and breeding.

Pollen PCP-B peptides unlock a stigma peptide–receptor kinase gating mechanism for pollination

Sexual reproduction in angiosperms relies on precise communications between the pollen and pistil. The molecular mechanisms underlying these communications remain elusive. We established that in Arabidopsis, a stigmatic gatekeeper, the ANJEA–FERONIA (ANJ–FER) receptor kinase complex, perceives the RAPID ALKALINIZATION FACTOR peptides RALF23 and RALF33 to induce reactive oxygen species (ROS) production in the stigma papillae, whereas pollination reduces stigmatic ROS, allowing pollen hydration. Upon pollination, the POLLEN COAT PROTEIN B-class peptides (PCP-Bs) compete with RALF23/33 for binding to the ANJ–FER complex, leading to a decline of stigmatic ROS that facilitates pollen hydration. Our results elucidate a molecular gating mechanism in which distinct peptide classes from pollen compete with stigma peptides for interaction with a stigmatic receptor kinase complex, allowing the pollen to hydrate and germinate.

Family-wide evaluation of RALF peptides in Arabidopsis thaliana

Plant peptide hormones are important players controlling various aspects of plants’ lives. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies on RALF peptides focused on broad phylogenetic analysis including multiple species. Thus, progress has been made in understanding the evolutionary processes that shaped this family. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analysed the phylogeny and function of the proposed multigenic RALF peptide family in the model plant Arabidopsis thaliana, ecotype Col-0. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a new consensus AtRALF peptide family annotation. Moreover, we show that the majority of AtRALF peptides are able to induce seedling or root growth inhibition in A. thaliana seedlings when applied exogenously as synthetic peptides. Additionally, we show that most of these responses are dependent on the Catharanthus roseus RLK1-LIKE receptor kinase FERONIA, suggesting a pivotal role in the perception of multiple RALF peptides.One sentence summaryThe majority of A. thaliana RALF peptides inhibit growth in a FERONIA-dependent manner

RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants

The environmentally responsive signaling pathways that link global transcriptomic changes through alternative splicing (AS) to plant fitness remain unclear. Here, we found that the interaction of the extracellular rapid alkalinization FACTOR 1 (RALF1) peptide with its receptor FERONIA (FER) triggered a rapid and massive RNA AS response by interacting with and phosphorylating glycine-rich RNA binding protein7 (GRP7) to elevate GRP7 nuclear accumulation in Arabidopsis thaliana. FER-dependent GRP7 phosphorylation enhanced its mRNA binding ability and its association with the spliceosome component U1-70K to enable splice site selection, modulating dynamic AS. Genetic reversal of a RALF1-FER–dependent splicing target partly rescued mutants deficient in GRP7. AS of GRP7 itself induced nonsense-mediated decay feedback to the RALF1-FER-GRP7 module, fine-tuning stress responses, and cell growth. The RALF1-FER-GRP7 module provides a paradigm for regulatory mechanisms of RNA splicing in response to external stimuli.

The Genome-Wide Analysis of RALF-Like Genes in Strawberry (Wild and Cultivated) and Five Other Plant Species (Rosaceae)

The rapid alkalinization factor (RALF) gene family is essential for the plant growth and development. However, there is little known about these genes among Rosaceae species. Here, we identify 124 RALF-like genes from seven Rosaceae species, and 39 genes from Arabidopsis, totally 163 genes, divided into four clades according to the phylogenetic analysis, which includes 45 mature RALF genes from Rosaceae species. The YISY motif and RRXL cleavage site are typical features of true RALF genes, but some variants were detected in our study, such as YISP, YIST, NISY, YINY, YIGY, YVGY, FIGY, YIAY, and RRVM. Motif1 is widely distributed among all the clades. According to screening of cis-regulatory elements, GO annotation, expression sequence tags (EST), RNA-seq, and RT-qPCR, we reported that 24 RALF genes coding mature proteins related to tissue development, fungal infection, and hormone response. Purifying selection may play an important role in the evolutionary process of RALF-like genes among Rosaceae species according to the result from ka/ks. The tandem duplication event just occurs in four gene pairs (Fv-RALF9 and Fv-RALF10, Md-RALF7 and Md-RALF8, Pm-RALF2 and Pm-RALF8, and Pp-RALF11 and Pp-RALF14) from four Rosaceae species. Our research provides a wide overview of RALF-like genes in seven Rosaceae species involved in identification, classification, structure, expression, and evolution analysis.

Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals, starting from root elongation, cell growth, pollen tube development and fertilization. Their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF peptides are secreted by different fungal pathogens as effectors during early stages of infections. Campbell and Turner previously identified nine RALF genes in F. vesca v1 genome. Here, based on the recent release of Fragaria x ananassa genome and F. vesca reannotation, we aimed to characterize the genomic organization of the RALF gene family in both type of strawberry species according to tissue specific expression and homology with Arabidopsis. We reveal the presence of 13 RALF genes in F. vesca and 50 in Fragaria x ananassa, showing a non-homogenous localization of genes among the different Fragaria x ananassa subgenomes associated with their different TE element contents and genome remodeling during evolution. Fragaria x ananassa RALF genes expression inducibility upon infection with C. acutatum or B. cinerea was assessed and showed that, among fruit expressed RALF genes, FaRALF3-1 was the only one upregulated after fungal infection. In silico analysis and motif frequency analysis of the putative regulatory elements upstream of the FaRALF3 gene was carried out in order to identify distinct pathogen inducible elements. Agroinfiltration of strawberry fruit with 5’ deletion constructs of the FaRALF3-1 promoter identified a region required for FaRALF3 expression in fruit, but did not identify a region responsible for fungal induced expression.

Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis

The perception and relay of cell-wall signals are critical for plants to regulate growth and stress responses, but the underlying mechanisms are poorly understood. We found that the cell-wall leucine-rich repeat extensins (LRX) 3/4/5 are critical for plant salt tolerance in Arabidopsis. The LRXs physically associate with the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23, which in turn interact with the plasma membrane-localized receptor-like protein kinase FERONIA (FER). The lrx345 triple mutant as well as fer mutant plants display retarded growth and salt hypersensitivity, which are mimicked by overexpression of RALF22/23. Salt stress promotes S1P protease-dependent release of mature RALF22 peptides. Treatment of roots with mature RALF22/23 peptides or salt stress causes the internalization of FER. Our results suggest that the LRXs, RALFs, and FER function as a module to transduce cell-wall signals to regulate plant growth and salt stress tolerance.